Methods for spatial analysis using rolling circle amplification and detection probes

ABSTRACT

Provided herein are methods of improving sensitivity of spatial detection of an analyte in a biological sample using splint oligonucleotides, circularized second strands, and rolling circle amplification. For example, provided herein are methods of improving sensitivity of spatial detection of an analyte in a biological sample where a splint oligonucleotide hybridizes to a second strand; the second strand is ligated together thereby creating a circularized second strand, rolling circle amplification of the circularized second strand results in generation of an amplified second strand, and all or part of the sequence of the amplified second strand is determined and used to spatially detect the analyte in the biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 63/115,916, filed Nov. 19, 2020, the entire contents of which areincorporated by reference herein.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provides substantial analytedata for dissociated tissue (i.e., single cells), but fail to provideinformation regarding the position of the single cell in a parentbiological sample (e.g., tissue sample).

Generally, spatial analysis requires determining the sequence of theanalyte sequence or a complement thereof and the sequence of the spatialbarcode or a complement thereof in order to identify spatial location ofthe analyte. Typically, this requires sequencing which can be time andresource intensive. Therefore, there is a need to assess analyte andbiological sample quality prior to spatial analysis.

SUMMARY

Spatial analysis requires processing of a captured analyte in order todetermine its abundance and location in a biological sample. During thisprocess, after capture of the analyte, a process of second strandsynthesis is performed. This process includes generating asingle-stranded nucleic acid that is complementary both to the captureprobe and to the analyte (or a complement thereof). After, the secondstrand can be further processed, placed in a library, and sequenced. Thedisclosure has identified that sensitivity can be lost during secondstrand synthesis. To address this issue, the disclosure provides methodsof generating an amplified product comprising the capture probe orcomplement thereof and analyte or complement thereof using a splintoligonucleotide and rolling circle amplification—all performed on asubstrate.

In one embodiment, disclosed herein is a method of determining locationand abundance of an analyte in a biological sample. In some instances,the method includes: (a) hybridizing the analyte to a capture probe onan array, wherein the capture probe comprises a spatial barcode and acapture domain; (b) extending the capture probe using the analyte as atemplate, thereby generating an extended capture probe, and generating asecond strand comprising a sequence that is complementary to (i) theanalyte or a complement thereof and (ii) the spatial barcode or acomplement thereof; (c) denaturing the second strand from the extendedcapture probe under conditions wherein a 5′ end of the second strand anda 3′ end of the second strand dehybridize from the extended captureprobe; (d) hybridizing a splint oligonucleotide both to the 5′ end ofthe second strand and to the 3′ end of the second strand; (e) generatinga circularized second strand; (f) amplifying the circularized secondstrand, thereby creating an amplified second strand; and (g) determiningall or part of the sequence of the amplified second strand todetermining the location and the abundance of the analyte in thebiological sample.

In some instances, the analyte comprises a capture domain capturesequence that hybridizes to the capture domain, and wherein the capturedomain comprises a poly(T) sequence. In some instances, the capturedomain capture sequence comprises a poly(A) sequence. In some instances,the array comprises a plurality of capture probes.

In some instances, extending the capture probe and generating a secondstrand utilize a polymerase or reverse transcriptase. In some instances,denaturing comprises increasing the temperature, thereby dehybridizingthe 5′ end of the second strand and the 3′ end of the second strand fromthe extended capture probe. In some instances, before step (e) above,the method includes extending the 5′ end of the second strand using thesplint oligonucleotide as a template, thereby creating an extended 5′end. In some instances, generating the circularized second strandcomprises ligating the extended second portion to the first portionusing a ligase. In some instances, the ligase is a T4 DNA ligase.

In some instances, amplifying the circularized second strand comprisesrolling circle amplification (RCA) using the circularized second strandas a template. In some instances, the amplified second strand comprises(i) the spatial barcode or complement thereof and (ii) all or part ofthe analyte or a complement thereof. In some instances, the methodfurther includes hybridizing an oligonucleotide to a portion of theamplified second strand, thereby producing a double-stranded sequence,and wherein the double-stranded sequence comprises a restriction site.In some instances, the method further includes digesting thedouble-stranded sequence using a restriction enzyme.

In some instances, the splint oligonucleotide comprises a first sequencethat is substantially complementary to the 5′ end of the second strand,and a second sequence that is substantially complementary to the 3′ endof the second strand. In some instances, the capture probe furthercomprises one or more functional domains, a unique molecular identifier,a cleavage domain, and combinations thereof. In some instances, themethod further includes phosphorylating the 5′ end of the splintoligonucleotide prior to the ligation step; and/or phosphorylating the5′ end of the first portion of the second strand. In some instances, theamplifying step comprises hybridizing one or more amplification primersto the circularized second strand, and amplifying the circularizedsecond strand with a polymerase.

In some instances, the determining step comprises sequencing all or partof the sequence of the amplified second strand to determining thelocation and the abundance of the analyte in the biological sample.

In some instances, the biological sample comprises a formalin-fixed,paraffin-embedded (FFPE) tissue sample, a fresh tissue sample, or afrozen tissue sample.

Also disclosed herein are kits. In some instances, the kits include (a)an array comprising a plurality of capture probes, wherein a captureprobe of the plurality of capture probes comprises (i) a capture domainthat hybridizes to an analyte of a biological sample and (ii) a spatialbarcode; (b) one or more splint oligonucleotides and a ligase; (c) oneor more RCA primers and a Phi29 DNA polymerase; (d) one or morerestriction enzymes; and (e) instructions for performing any of themethods disclosed herein.

Also disclosed herein is a method of spatially detecting of an analytein a biological sample comprising: (a) hybridizing the analyte to acapture probe comprising a spatial barcode and creating a second strandcomprising a sequence that is complementary to a portion of the analyteand an extended capture probe; (b) denaturing the second strand underconditions wherein a first portion of the second strand and a secondportion of the second strand de-hybridize from the extended captureprobe; (c) hybridizing a splint oligonucleotide to the first portion andto the second portion; (d) ligating part of the splint oligonucleotide,to the first portion, and the second portion thereby creating acircularized second strand; (e) amplifying the circularized secondstrand, thereby creating an amplified second strand; and (f) determiningall or part of the sequence of the amplified second strand to spatiallydetect the analyte in the biological sample.

Also disclosed herein is a method of spatially detecting of an analytein a biological sample comprising: (a) hybridizing the analyte to acapture probe comprising a spatial barcode and creating a second strandcomprising a sequence that is complementary to a portion of the analyteand an extended capture probe; (b) denaturing the second strand underconditions wherein a first portion of the second strand and a secondportion of the second strand de-hybridize from the extended captureprobe; (c) hybridizing a splint oligonucleotide to the first portion andto the second portion; (d) ligating part of the splint oligonucleotide,to the first portion, and the second portion thereby creating acircularized second strand; (e) amplifying the circularized secondstrand, thereby creating an amplified second strand; and (f) determiningall or part of the sequence of the amplified second strand to spatiallydetect the analyte in the biological sample.

In some instances, the capture probe is on a substrate comprising aplurality of capture probes, wherein the capture probe comprises acapture domain and the spatial barcode, and wherein the analytecomprises a capture probe binding domain that is capable of binding tothe capture domain. In some instances, the splint oligonucleotidecomprises a backbone sequence, wherein the backbone sequence comprises adouble-stranded sequence. In some instances, the methods further includeextending the second portion using the splint oligonucleotide as atemplate, thereby creating an extended second portion. In someinstances, the methods further include ligating the extended secondportion to the first portion, thereby creating a circularized secondstrand. In some instances, the methods further include extending thecapture probe, thereby creating an extended capture probe. In someinstances, the methods further include amplifying the extended captureprobe creating a second strand comprising a sequence that iscomplementary to a portion of the analyte and the extended captureprobe. In some instances, the determined sequence of the amplifiedsecond strand comprises the spatial barcode or complementary sequencethereof for spatially detecting the analyte in the biological sample. Insome instances, the splint oligonucleotide comprises a first sequencethat is substantially complementary to the second portion, and a secondsequence that is substantially complementary to the first portion. Insome instances, the methods further include digesting the amplifiedsecond strand. In some instances, the method improves sensitivity ofspatial detection of an analyte as compared to methods of spatiallydetecting an analyte that does not include rolling circle amplification.

Also disclosed herein is a method of spatially detecting of an analytein a biological sample comprising: (a) hybridizing the analyte to acapture probe comprising a spatial barcode and extending the captureprobe, thereby creating an extended capture probe; (b) amplifying theextended capture probe creating a second strand comprising a sequencethat is complementary to a portion of the analyte and the extendedcapture probe; (c) hybridizing a splint oligonucleotide to a firstportion of the second strand and a second portion of the second strand,(d) ligating part of the splint oligonucleotide, or a complementthereof, to the first portion and the second portion creating acircularized second strand; (e) amplifying the circularized secondstrand creating an amplified second strand and digesting the amplifiedsecond strand, thereby producing a plurality of second strand fragments;and (f) determining all or part of the sequence of a second strandfragment, and using the determined sequence to spatially detect theanalyte in the biological sample. In some instances, the capture probeis on a substrate comprising a plurality of capture probes, wherein thecapture probe comprises a capture domain and the spatial barcode, andwherein the analyte comprises a capture probe binding domain that iscapable of binding to the capture domain. In some instances, the splintoligonucleotide comprises a backbone sequence, wherein the backbonesequence comprises a double-stranded sequence. In some instances, themethods further include denaturing the second strand under conditionswherein a first portion of the second strand and a second portion of thesecond strand de-hybridize from the extended capture probe. In someinstances, the splint oligonucleotide comprises a first sequence that issubstantially complementary to the second portion, and a second sequencethat is substantially complementary to the first portion. In someinstances, the determined sequence of the second strand fragmentcomprises the spatial barcode of the capture probe or a complementarysequence thereof.

In some instances, digesting comprises inducing a plurality ofdouble-stranded breaks in the amplified second strand, wherein adouble-stranded break occurs at a specific nucleic acid position(s) inthe amplified second strand. In some instances, digesting comprises:hybridizing an oligonucleotide to a portion of the amplified secondstrand, thereby producing a double-stranded sequence, wherein thedouble-stranded sequence comprises a restriction site; and digesting thedouble-stranded sequence using a restriction enzyme. In some instances,the restriction site is added to the circularized second strand duringthe amplifying step. In some instances, the amplifying step comprisesrolling circle amplification using the circularized second strand as atemplate. In some instances, a second strand fragment of the pluralityof second strand fragments comprises all or part of the analyte orcomplement thereof and a spatial barcode or a complement thereof.

In some instances, the methods further include amplifying the secondstrand fragment prior to determining the sequence of the second strandfragment, thereby generating an amplified second strand fragment. Insome instances, the methods further include determining the sequence ofthe amplified second strand fragment, wherein the determined sequence ofthe amplified second strand fragment comprises the spatial barcodesequence of the capture probe or a complementary sequence thereof, andusing the determined sequence of the amplified second strand fragment orthe spatial barcode to spatially detect the analyte in the biologicalsample. In some instances, the amplifying and digesting steps areperformed concurrently.

In some instances, the oligonucleotide comprises a blocking moiety onthe 3′ end.

In some instances, the method improves sensitivity of spatial detectionof an analyte as compared to methods of spatially detecting an analytethat do not include any method disclosed herein.

In some instances, the second portion of the second strand comprises oneor more of a spatial barcode, a unique molecular identifier, and aprimer sequence. In some instances, the second portion of the secondstrand comprises a portion of the captured analyte. In some instances,the first portion of the second strand comprises a primer sequence. Insome instances, the first portion of the second strand comprises aportion of the captured analyte. In some instances, the splintoligonucleotide further comprises one or more of a functional sequenceand a unique barcode. In some instances, the functional sequence is aprimer sequence. In some instances, the primer is used for amplifyingthe circularized second strand.

In some instances, the ligating step comprises a T4 DNA ligase.

In some instances, the methods further include phosphorylating the 5′end of the double-stranded splint backbone sequence of the splintoligonucleotide prior to the ligation step. In some instances, themethods further include phosphorylating the 5′ end of the first portionof the second strand.

In some instances, the capture domain comprises a sequence that is atleast partially complementary to the analyte. In some instances, thecapture domain of the capture probe comprises a homopolymeric sequence.In some instances, the capture domain of the capture probe comprises apoly(T) sequence. In some instances, the capture domain of the captureprobe comprises a non-homopolymeric sequence. In some instances, thenon-homopolymeric sequence is a random sequence, a partially randomsequence or a fully defined sequence. In some instances, the captureprobe comprises a cleavage domain. In some instances, the cleavagedomain comprises a cleavable linker selected from a photocleavablelinker, a UV-cleavable linker, an enzyme-cleavable linker, or apH-sensitive cleavable linker. In some instances, extending the captureprobe comprises reverse transcribing the analyte or complementarysequence thereof. In some instances, extending the capture probecomprises generating a sequence that is complementary to a portion ofthe analyte. In some instances, reverse transcribing the analytegenerates a reverse complement of a template switching oligonucleotide.In some instances, amplifying the extended capture probe comprisesannealing a template switching oligonucleotide primer to the reversecomplement of the template switching oligonucleotide. In some instances,amplifying further comprises hybridizing the 3′ end of the extendedcapture probe to the first portion of the second strand, and using the3′ end of the extended capture probe as a substrate in a rolling circleamplification reaction.

In some instances, the amplifying step (h) comprises: hybridizing one ormore amplification primers to the circularized second strand, thecapture probe, or the analyte; and amplifying the circularized secondstrand with a polymerase. In some instances, the determining stepcomprises sequencing. In some instances, the sequencing comprisesgenerating a sequencing library of the amplified second strand, secondstrand fragments, or amplified second strand fragments.

In some instances, the biological sample comprises a FFPE sample. Insome instances, the biological sample comprises a tissue section. Insome instances, the biological sample comprises a fresh frozen sample.In some instances, the biological sample comprises live cells.

Also disclosed herein is a composition comprising the circularizedsecond strand, wherein the circularized second strand comprises a firstportion of the second strand, a portion of the second strand thatremained hybridized to the extended capture probe, a second portion ofthe second strand, and a portion of the backbone sequence of the splintoligonucleotide.

Also disclosed herein is a kit, comprising: a substrate for spatialdetection of an analyte, one or more splint oligonucleotides and aligase; one or more RCA primers and a Phi29 DNA polymerase; andinstructions for performing any of the methods disclosed herein.

Also disclosed herein is a kit, comprising: a substrate for spatialdetection of an analyte; one or more splint oligonucleotides and aligase; one or more RCA primers and a Phi29 DNA polymerase; one or moreoligonucleotides and one or more restriction enzymes; and instructionsfor performing any of the methods disclosed herein.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, patent application, or item ofinformation was specifically and individually indicated to beincorporated by reference. To the extent publications, patents, patentapplications, and items of information incorporated by referencecontradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 2 is a schematic illustrating exemplary, non-limiting,non-exhaustive steps for second strand synthesis.

FIG. 3A is a schematic illustrating exemplary, non-limiting,non-exhaustive steps for generating a circularized second strand.

FIG. 3B is a schematic illustrating exemplary, non-limiting,non-exhaustive steps for amplifying a circularized second strand.

FIG. 4 is a schematic illustrating exemplary, non-limiting,non-exhaustive steps for digesting an amplified second strand usingoligonucleotides, partially double stranded amplified second strands,and a restriction enzyme.

DETAILED DESCRIPTION

Disclosed herein are methods of improving sensitivity of spatialdetection of an analyte in a biological sample. The techniques disclosedherein facilitate downstream processing by increasing the abundance ofan analyte (e.g., increasing copies of an analyte or derivativesthereof) from a biological sample. For example, an analyte or an analytederived molecule (e.g., a second strand cDNA molecule) is amplifiedprior to determining all or part of the sequence of the analyte. Theamplification method described herein amplifies a spatial barcode of acapture probe or a complement thereof in addition to all or part of thesequence of an analyte or a complement thereof. This enables retentionof spatial information. A splint oligonucleotide is hybridized to afirst portion and a second portion of a second strand (e.g., a secondstrand cDNA molecule), where the second strand includes the spatialbarcode or a complement thereof and all or part of the sequence of theanalyte or a complement thereof. In some cases, hybridization of thesplint oligonucleotide to the first portion and the second portion isenabled by denaturing the second strand hybridized to the extendedcapture probe under conditions that allow the first portion and thesecond portion to de-hybridize from the extended capture probe. Thesplint oligonucleotide mediates ligation of the second portion to thefirst portion of the second strand thereby producing a circularizedsecond strand. The circularized second strand is amplified therebycreating an amplified second stand that includes the spatial informationnecessary (e.g., information including the sequence the analyte or acomplement thereof and the sequence of spatial barcode or a complementthereof) to spatially detect the analyte in the biological sample.“Improving sensitivity” of spatial detection as used herein refers to anincreased detection of an analyte at a location of a sample usingmethods disclosed herein compared to a reference sample.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination, and each of which isincorporated herein by reference in their entireties. Furthernon-limiting aspects of spatial analysis methodologies and compositionsare described herein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Generation of capture probes can be achieved by any appropriate method,including those described in Section (II)(d)(ii) of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

FIG. 1 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that are usefulfor subsequent processing. The functional sequence 104 can include allor part of sequencer specific flow cell attachment sequence (e.g., a P5or P7 sequence), all or part of a sequencing primer sequence, (e.g., aR1 primer binding site, a R2 primer binding site), or combinationsthereof. The capture probe can also include a spatial barcode 105. Thecapture probe can also include a unique molecular identifier (UMI)sequence 106. While FIG. 1 shows the spatial barcode 105 as beinglocated upstream (5′) of UMI sequence 106, it is to be understood thatcapture probes wherein UMI sequence 106 is located upstream (5′) of thespatial barcode 105 is also suitable for use in any of the methodsdescribed herein. The capture probe can also include a capture domain107 to facilitate capture of a target analyte. In some embodiments, thecapture probe comprises one or more additional functional sequences thatcan be located, for example between the spatial barcode 105 and the UMIsequence 106, between the UMI sequence 106 and the capture domain 107,or following the capture domain 107. The capture domain can have asequence complementary to a sequence of a nucleic acid analyte. Thecapture domain can have a sequence complementary to a connected probedescribed herein. The capture domain can have a sequence complementaryto a capture handle sequence present in an analyte capture agent. Thecapture domain can have a sequence complementary to a splintoligonucleotide. Such splint oligonucleotide, in addition to having asequence complementary to a capture domain of a capture probe, can havea sequence of a nucleic acid analyte, a sequence complementary to aportion of a connected probe described herein, and/or a capture handlesequence described herein.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. As used herein, the term “analyte binding moietybarcode” refers to a barcode that is associated with or otherwiseidentifies the analyte binding moiety. As used herein, the term “analytecapture sequence” refers to a region or moiety configured to hybridizeto, bind to, couple to, or otherwise interact with a capture domain of acapture probe. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent. Additional description of analyte capture agentscan be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section(II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g.,Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663 regarding extended capture probes). In somecases, capture probes may be configured to form ligation products with atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent, or portion thereof), thereby creating ligationsproducts that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder. Exemplary methods for identifying spatial information ofbiological and/or medical importance can be found in U.S. PatentApplication Publication No. 2021/0140982A1, U.S. Patent Application No.2021/0198741A1, and/or U.S. Patent Application No. 2021/0199660.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in WO 2021/102003and/or U.S. patent application Ser. No. 16/951,854, each of which isincorporated herein by reference in their entireties.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2021/102039and/or U.S. patent application Ser. No. 16/951,864, each of which isincorporated herein by reference in their entireties.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, WO2021/102005, and/or U.S. patent application Ser. No. 16/951,843, each ofwhich is incorporated herein by reference in their entireties. Fiducialmarkers can be used as a point of reference or measurement scale foralignment (e.g., to align a sample and an array, to align twosubstrates, to determine a location of a sample or array on a substraterelative to a fiducial marker) and/or for quantitative measurements ofsizes and/or distances.

Surface Amplification on a Substrate to Improve Sensitivity of SpatialDetection

This disclosure features methods of improving sensitivity of spatialdetection of an analyte in a biological sample. In other methods ofspatial analysis, after capture of an analyte on a capture probe affixedto an array, a second strand, which is complementary to the captureprobe and to all or part of the analyte, is generated, followed by cDNAamplification, library preparation of the amplified secondary strand,and sequencing. The methods provided herein disclose an alternativemethod of spatial detection. In part, disclosed herein are methods usinga splint oligonucleotide that can bind to a second strand (e.g., asecond strand cDNA molecule bound to an analyte that is bound to acapture probe) and enable ligation of the second portion to the firstportion thereby creating a circularized second strand. The circularizedsecond strand can be amplified, all or part of the sequence of theamplified second strand can be determined, and the determined sequenceused to spatially detect the analyte in the biological sample. Thisdisclosure also features methods of improving sensitivity of spatialdetection of an analyte in a biological sample using fragmentation(e.g., digestion) of the amplified second strand. In a non-limitingexample, the method includes digesting the amplified second strandthereby generating second strand fragments, and determining all or partof the sequence of the second strand fragments to spatially detect theanalyte in the biological sample.

This disclosure also features a method of improving sensitivity ofspatial detection of an analyte in a biological sample where the methodincludes: providing a biological sample including an analyte on asubstrate, wherein the substrate includes a plurality of capture probescomprising capture domains that are capable of hybridizing to an analytesequence; hybridizing the analyte to the capture domain and creating asecond strand including a sequence that is complementary to a portion ofthe analyte and the extended capture probe; hybridizing a splintoligonucleotide to a first portion of the second strand and to a secondportion of the second strand; ligating part of the splintoligonucleotide, to the first portion, and the second portion therebycreating a circularized second strand; amplifying the circularizedsecond strand, thereby creating an amplified second strand; anddetermining all or part of the sequence of the amplified second strandto spatially locate the analyte in the biological sample. In someembodiments, the method further includes extending the capture probecreating an extended capture probe. In some embodiments, the methodfurther includes amplifying the extended capture probe creating a secondstrand including a sequence that is complementary to a portion of theanalyte and the extended capture probe. In some embodiments, the methodfurther includes subjecting the second strand to a denaturing step underconditions wherein the first portion of the second strand and the secondportion of the second strand de-hybridize from the extended captureprobe.

This disclosure also features a method of improving sensitivity ofspatial detection of an analyte in a biological sample where the methodincludes: a biological sample including an analyte on a substratewherein the substrate includes a plurality of capture probes comprisinga capture domain, and wherein the analyte includes a capture probebinding domain that is capable of binding to the capture domain;hybridizing the capture probe binding domain to the capture domain andcreating a second strand including a sequence that is complementary to aportion of the analyte and the extended capture probe; denaturing thesecond strand under conditions wherein a first portion of the secondstrand and a second portion of the second strand de-hybridize from theextended capture probe; hybridizing a splint oligonucleotide to thefirst portion and to the second portion; ligating part of the splintoligonucleotide, to the first portion, and the second portion therebycreating a circularized second strand; amplifying the circularizedsecond strand, thereby creating an amplified second strand; anddetermining all or part of the sequence of the amplified second strandto spatially detect the analyte in the biological sample. In someembodiments, the method further includes extending the capture probecreating an extended capture probe. In some embodiments, the methodfurther includes amplifying the extended capture probe creating a secondstrand including a sequence that is complementary to a portion of theanalyte and the extended capture probe.

In some embodiments, the method further includes digesting the amplifiedsecond strand. In some embodiments, the amplifying step and thedigesting step are performed in one reaction. For example, the methodcan include a step concurrently amplifying the circularized secondstrand creating an amplified second strand and digesting the amplifiedsecond strand, thereby producing a plurality of second strand fragments.

In a non-limiting example, this disclosure features a method improvingsensitivity of spatial detection of an analyte in a biological samplewhere the method includes: a biological sample including an analyte on asubstrate, wherein the substrate includes a plurality of capture probescomprising a capture domain, and wherein the analyte includes a captureprobe binding domain that is capable of binding to the capture domain;hybridizing the capture probe binding domain to the capture domain andextending the capture probe creating an extended capture probe;amplifying the extended capture probe creating a second strand includinga sequence that is complementary to a portion of the analyte and theextended capture probe; hybridizing a splint oligonucleotide to a firstportion of the second strand and to a second portion of the secondstrand, ligating part of the splint oligonucleotide, or a complementthereof, to the first portion and the second portion creating acircularized second strand; concurrently amplifying the circularizedsecond strand creating an amplified second strand and digesting theamplified second strand, thereby producing a plurality of second strandfragments; and determining all or part of the sequence of a secondstrand fragment, and using the determined sequence to spatially detectthe analyte in the biological sample. In some embodiments, the methodfurther includes subjecting the second strand to a denaturing step underconditions wherein a first portion of the second strand and a secondportion of the second strand de-hybridize from the extended captureprobe.

(a) Splint Oligonucleotide and Circularization of the Second Strand

This disclosure features methods for improving sensitivity of spatialdetection of an analyte in a biological sample using a splintoligonucleotide hybridized to a second strand. In some embodiments, themethod includes ligating part of the splint oligonucleotide to a firstportion of the second strand and to a second portion of the secondstrand, thereby creating a circularized second strand. As used herein, a“splint oligonucleotide” refers to an oligonucleotide that has, at its5′ and 3′ ends, sequences (e.g., a first sequence at the 5′ end and asecond sequence at the 3′ end) that are complementary to portions (e.g.,a first portion and a second portion) of the second strand. Uponhybridization to the first and second portions of the second strand, thesplint oligonucleotide brings the two portions of the second strand intocontact, allowing circularization of the second strand by ligation(e.g., ligation using any of the methods described herein). Uponhybridization to the first and second portions of the second strand, thesecond portion of the second strand is extended until the two portionsof the second strand are brought into contact, allowing circularizationof the second strand by ligation (e.g., ligation using any of themethods described herein). The ligation product can be referred to asthe “circularized second strand.” In some embodiments, aftercircularization of the second strand, rolling circle amplification canbe used to amplify the circularized second strand creating an “amplifiedsecond strand.”

In some embodiments, a first sequence of a splint oligonucleotideincludes a sequence that is substantially complementary to a secondportion of the second strand. In some embodiments, the second portion ofthe second strand is 3′ to the first portion of the second strand. Insome embodiments, the first sequence is at least 70% identical (e.g., atleast 75% identical, at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, or at least 99% identical)to the second portion.

In some embodiments, the splint oligonucleotide includes a backbonesequence. In some embodiments, the backbone sequence is double strandedwhile the first sequence and second sequences are single stranded. Insome embodiments, the splint oligonucleotide is double stranded. In someembodiments, the backbone sequence includes a sequence that issubstantially complementary to an amplification primer. Theamplification primer can be a primer used in a rolling circleamplification reaction (RCA), where the RCA increases the “copy number”of the analyte and analyte derived molecules. In some embodiments, thebackbone sequence includes a functional sequence. In some embodiments,the backbone sequence includes a restriction site. For example, thebackbone sequence includes a sequence that is recognized by arestriction enzyme. In such cases, the backbone sequence can beconverted from a single stranded sequence to a double stranded sequence(e.g., a double stranded sequence that includes a functional restrictionsite) by adding an oligonucleotide that is substantially complementaryor by performing a nucleic acid extension reaction. In some embodiments,a double stranded backbone sequence includes a restriction site. In someinstances, the restriction site is not in the captured analyte. In someinstances, the restriction site is not in the genome of the biologicalsample.

In some embodiments, a second sequence of a splint oligonucleotideincludes a sequence that is substantially complementary to a firstportion of the second strand. In some embodiments, the first portion ofthe second strand is 3′ to the second portion of the second strand. Insome embodiments, the second sequence is at least 70% identical (e.g.,at least 75% identical, at least 80% identical, at least 85% identical,at least 90% identical, at least 95% identical, or at least 99%identical) to the first portion.

In some embodiments, the splint oligonucleotide does not include abackbone sequence. In such cases, the first sequence and the secondsequence are directly adjacent to each other on the splintoligonucleotide. As such, when the splint oligonucleotide is hybridizedto the second strand, the first portion of the second strand and thesecond portion of the second strand hybridize to adjacent sequences onthe splint oligonucleotide. This enables ligation without having toperform a gap filling step.

In some embodiments, the splint oligonucleotide includes a backbonesequence where the first sequence is not directly adjacent to the secondsequence. In such cases, a “gap” exists on the splint oligonucleotidebetween where the first sequence is hybridized to the second portion andwhere the second sequence is hybridized to the first portion. In someembodiments, the splint oligonucleotide includes a sequence (e.g., agap) between the first sequence and the second sequence of at least1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8,1-7, 1-6, 1-5, 1-4, 1-3, 1-2 or 1 nucleotide(s). In a non-limitingexample, a first sequence having a sequence that is substantiallycomplementary to a second portion of the second strand and a secondsequence having a sequence that is substantially complementary to afirst portion of the second strand each bind to the second strandleaving a sequence (e.g., the “gap”) in between the first and secondsequences that is gap-filled thereby enabling ligation and generation ofthe circularized second strand. In some instances, to generate a splintoligonucleotide that includes a first sequence and a second sequencethat are close enough to one another to initiate a ligation step, thesecond sequence is extended enzymatically (e.g., using a reversetranscriptase).

In some embodiments, the “gap” sequence between the first sequence andthe second sequence include one nucleotide, two nucleotides, threenucleotides, four nucleotides, five nucleotides, six nucleotides, sevennucleotides, eight nucleotides, nine nucleotides, ten nucleotides, 11nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23nucleotides, 24 nucleotides, at least 25 nucleotides, at least 30nucleotide, at least nucleotides, at least 40 nucleotides, at least 45nucleotides, or at least 50 nucleotides.

In some embodiments, the gap is filled by extending the second portionof the second strand. In some embodiments, extending the second portionof the second strand includes a nucleic acid extension reaction (e.g.,any of the nucleic acid extension reactions described herein). In someembodiments, extending the second portion of the second strand includesreverse transcribing the splint oligonucleotide. In some embodiments,extending the second portion of the second strand includes using areverse transcriptase (e.g., any of the reverse transcriptases describedherein). In some embodiments, extending the second portion of the secondstrand includes using a Moloney Murine Leukemia Virus (M-MulV) reversetranscriptase. In some embodiments, the reverse transcriptase includesstrand displacement properties. In some embodiments, extending thesecond portion of the second strand generates a sequence that iscomplementary to the splint oligonucleotide. In some embodiments,extending the second portion of the second strand generates an extendedsecond sequence of the second strand that is complementary to the splintoligonucleotide. In some embodiments, extending the second portion ofthe second strand generates a sequence that is adjacent to the firstportion of the second strand.

In some embodiments, the ligation step includes ligating the secondportion to the first portion of the second strand using enzymatic orchemical ligation. In some embodiments where the ligation is enzymatic,the ligase is selected from a T4 RNA ligase (Rnl2), a SplintR ligase, asingle stranded DNA ligase, or a T4 DNA ligase. In some embodiments, theligase is a T4 RNA ligase (Rn12) ligase. In some embodiments, the ligaseis a pre-activated T4 DNA ligase as described herein. A non-limitingexample describing methods of generating and using pre-activated T4 DNAinclude U.S. Pat. No. 8,790,873, the entire contents of which are hereinincorporated by reference.

(b) Amplification of Circularized Second Strand

This disclosure features methods for improving sensitivity of spatialdetection of an analyte in a biological sample by amplifying thecircularized second strand. In a non-limiting example, the methodincludes an amplifying step wherein one or more amplification primersare hybridized to the circularized second strand and the circularizedsecond strand is amplified using a polymerase. In another non-limitingexample, the method includes an amplifying step where a 3′ end of theextended capture probe is used as a primer and the circularized secondstrand is amplified using a polymerase. In some embodiments, theamplifying step increases the copy number of the second strand. Thesequence of the amplified second strand can then be determined and usedto spatially detect the analyte in the biological sample. In someembodiments, the amplifying step includes rolling circle amplification(RCA).

As used herein, rolling circle amplification (RCA) can refer to apolymerization reaction carried out using a single-stranded circular DNA(e.g., a circularized second strand) as a template and an amplificationprimer that is substantially complementary to the single-strandedcircular DNA (e.g., the circularized second strand) to synthesizemultiple continuous single-stranded copies of the template DNA (e.g.,the circularized second strand). In some embodiments, RCA includeshybridizing one or more amplification primers to the circularized secondstrand and amplifying the circularized strand using a Phi29 DNApolymerase. In addition, RCA can refer to a polymerization reactioncarried out using a single-stranded circular DNA (e.g., a circularizedsecond strand) as a template and the 3′ end of the extended captureprobe as a primer to synthesize multiple continuous single-strandedcopies of the template DNA (e.g., the circularized second strand). Insuch cases, the 3′ end of the extended capture is substantiallycomplementary to a portion (e.g., a portion corresponding to the firstportion of the second strand) of the single-stranded circular DNA (e.g.,the circularized second strand). For example, RCA can includehybridizing the 3′ end of the extended capture probe to the circularizedsecond strand and amplifying the circularized strand using a Phi29 DNApolymerase.

In some embodiments, an amplification primer includes a sequence that issubstantially complementary to the first portion or the second portionof the circularized second strand. For example, the amplification primercan be substantially complementary to the first portion or a complementthereof. By substantially complementary, it is meant that theamplification primer is at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to a sequence in the circularizedsecond strand.

In some embodiments, the amplifying step includes hybridizing the 3′ endof the extended capture probe to the first portion of the second strand,and using the 3′ end of the extended capture probe as a substrate in arolling circle amplification (RCA) reaction. In such cases, the methodincludes a denaturing step under conditions where the 3′ end of theextended capture probe is de-hybridized from the first portion of thesecond strand. During the subsequent processing of the second strand(e.g., ligation and circularization) the second strand can remainhybridized to the extended capture probe. Therefore, the 3′ end of theextended capture probe can be re-hybridized to the first portion of thesecond strand.

In some embodiments, non-limiting examples of DNA polymerase include:Bsu DNA polymerase, Bst DNA polymerase, VENT™ DNA polymerase, DEEPVENT™DNA polymerase, Phi29 DNA polymerase, Klenow fragment, T4 DNA polymeraseand T7 DNA polymerase enzymes. In some embodiments, the DNA polymeraseis Phi29 DNA polymerase. In some embodiments, the term “DNA polymerase”includes not only naturally-occurring enzymes but also all modifiedderivatives thereof, including derivatives of naturally-occurring DNApolymerase enzymes. For instance, in some embodiments, the DNApolymerase is modified to remove 5′-3′ exonuclease activity.Sequence-modified derivatives or mutants of DNA polymerase enzymes thatcan be used include, but are not limited to, mutants that retain atleast some of the functional, e.g., DNA polymerase activity of thewild-type sequence.

(c) Capture Probe Extension, Second Strand Synthesis and De-hybridizing

This disclosure features methods for improving sensitivity of spatialdetection of an analyte in a biological sample using de-hybridization ofa second strand, hybridization of a splint oligonucleotide,circularization of the second strand, and amplification of acircularized second strand. In a non-limiting example, the methodincludes capturing an analyte, extending a capture probe, generating asecond strand, de-hybridizing a first and second portion of the secondstrand, hybridizing a splint oligonucleotide to the second strand,circularizing the second strand and amplifying the circularized secondstrand.

In some embodiments, extending the capture probe includes performing anucleic acid extension reaction. In some embodiments, extending thecapture probe includes reverse transcribing the analyte or complementarysequence thereof. In some embodiments, extending the capture probeincludes generating a sequence that is complementary to a portion of theanalyte. In some embodiments, extending the capture probe includesattaching a template switching oligonucleotide to the analyte. In someembodiments, reverse transcribing the analyte generates a reversecomplement of a template switching oligonucleotide.

In some embodiments, amplifying the extended capture probe includesannealing a template switching oligonucleotide (TSO) primer to a reversecomplement of the template switching oligonucleotide (rcTSO). In someembodiments, extending the capture probe and/or amplifying the extendedcapture probe includes using a polymerase. A non-limiting example of apolymerase is a DNA polymerase.

In some embodiments, extending the capture probe includes a reversetranscriptase enzyme, where the enzyme includes one or more of terminaltransferase activity, template switching ability, strand displacementability, or combinations thereof. In some embodiments, the terminaltransferase activity of the reverse transcriptase adds untemplatednucleotides to the 3′ end of the cDNA molecule. In some embodiments, thereverse transcriptase adds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreuntemplated nucleotides to the 3′ end of the cDNA molecule. In someembodiments the first enzyme includes a Moloney Murine Leukemia Virus(M-MLV) reverse transcriptase enzyme. In some embodiments the firstenzyme includes a Moloney Murine Leukemia Virus (M-MLV) reversetranscriptase enzyme and the second enzyme is a Bst DNA polymerase. Insome embodiments the first enzyme includes a Moloney Murine LeukemiaVirus (M-MLV) reverse transcriptase enzyme and the second enzyme is aPhi29 DNA polymerase.

In some embodiments, second strand synthesis is performed by a DNApolymerase selected from the group including, but not limited to: E.coliDNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNApolymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, LongAmp® TaqDNA polymerase, LongAmp® Hot Start Taq DNA polymerase, Crimson LongAmp®Taq DNA polymerase, Crimson Taq DNA polymerase, OneTaq® DNA polymerase,OneTaq® Quick-Load® DNA polymerase, Hemo KlenTaq® DNA polymerase,REDTaq® DNA polymerase, Phusion® DNA polymerase, Phusion® High-FidelityDNA polymerase, Platinum Pfx DNA polymerase, AccuPrime Pfx DNApolymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase,Pfu DNA polymerase, T4 DNA polymerase and T7 DNA polymerase enzymes. Insome embodiments, the second strand synthesis is a Phi29 DNA polymerase.In some embodiments, the second strand synthesis is performed by a BstDNA polymerase.

In some embodiments, the method includes contacting the analyte bound tothe capture probe with a composition that includes a template switchingoligonucleotide (TSO). In some embodiments, the TSO includes anuntemplated nucleotide region and a TSO primer region. In someembodiments, the length of a template switching oligonucleotide can beat least about 1, 2, 20, or 50 nucleotides or longer. In someembodiments, the length of a template switching oligonucleotide can beat most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides orlonger.

In some embodiments, the TSO primer region includes a sequence that isat least partially complementary to the TSO primer. In some embodiments,the TSO primer region includes a sequence that is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, ornucleotides in length. In some embodiments, the untemplated nucleotideregion includes a sequence that is at least partially complementary tothe untemplated nucleotides added on to the 3′ end of the extendedcapture probe. In some embodiments, the untemplated nucleotide regionincludes a sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenucleotides in length.

In some embodiments, the untemplated nucleotide region includes a seriesof G bases to complement the overhanging C bases at the 3′ end of a cDNAmolecule. In some embodiments, the series of G bases can include 1 Gbase, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 Gbases. In some embodiments, the hybridization region can include atleast one base in addition to at least one G base. In other embodiments,the hybridization can include bases that are not a G base. In someembodiments, the template region and hybridization region are separatedby a spacer. In some embodiments, the reverse complement of the TSO(rcTSO) is incorporated at the 3′ end of the cDNA molecule when the TSObinds to the untemplated nucleotides on the cDNA molecule and thereverse transcriptase reverse transcribes the TSO.

In some embodiments, the method includes contacting the analyte bound tothe capture probe with a composition that includes a TSO primer. In someembodiments, the TSO primer includes a sequence that is at leastpartially complementary to the rcTSO sequence. In some embodiments, theTSO primer includes a sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ornucleotides in length. In some embodiments, the TSO primer includes anyof the deoxyribonucleic acids, ribonucleic acids, modified nucleicacids, or any combination therein (e.g., any of the nucleotidederivatives or combinations thereof described herein). In someembodiments, the TSO primer includes RNA bases. In some embodiments, theTSO primer does not include RNA bases.

In some embodiments, the TSO primer is a single-stranded nucleic acidwhere the 3′ end is used as a chemical substrate for a nucleic acidpolymerase in a nucleic acid extension reaction. In some embodiments,the TSO primer is used as a chemical substrate for a second strandsynthesis where the extended capture probe is used as a template in anucleic acid extension reaction, where the second strand iscomplementary to all or a portion of the cDNA molecule and all or aportion of the capture probe. In some embodiments, the TSO primer and afirst enzyme (e.g., a reverse transcriptase with DNA polymerasefunctionality) are used in second strand synthesis, where the secondstrand synthesis occurs in the same reaction as the reversetranscription. In some embodiments, the TSO primer and a second enzyme(e.g., a DNA polymerase) are used in a second strand synthesis reaction,where the second strand synthesis occurs in the same reaction as thereverse transcription.

In some embodiments, the method includes contacting the analyte bound tothe capture probe with a composition that includes a TSO blockingmoiety. In some embodiments, the TSO blocking moiety is a nucleotidesequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or nucleotides in length. In someembodiments, the TSO blocking moiety is a nucleotide sequence that is atleast partially complementary to the TSO. In some embodiments, the TSOblocking moiety prohibits the TSO primer from interacting with thercTSO. For example, the TSO blocking moiety can bind to the TSO primerthereby inhibiting the TSO primer from interacting the rcTSO. In someembodiment the TSO blocking moiety is a nucleotide sequence that is atleast partially complementary to the rcTSO. In some embodiments, the TSOblocking moiety prohibits the rcTSO from interacting with the TSOprimer. For example, the TSO blocking moiety can bind to the rcTSOthereby inhibiting the rcTSO from interacting with the TSO primer.

This disclosure features methods of improving sensitivity of spatialdetection of an analyte in a biological sample where a second strand iscircularized by denaturing the second strand under conditions wherein afirst portion of the second strand and a second portion of the secondstrand de-hybridize from the extended capture probe and are hybridizedto a splint oligonucleotide, ligated (e.g., circularized), andamplified.

In some embodiments, denaturing includes temperature modulation. Forexample, a first portion and a second portion have predeterminedannealing temperatures based on the nucleotide composition (A, G, C, orT) within the known sequence. In some embodiments, the temperature ismodulated up to 5° C., up to 10° C., up to 15° C., up to 20° C., up to25° C., up to 30° C., or up to 35° C. above the predetermined annealingtemperature. In some embodiments, the temperature is modulated at 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35° C. above thepredetermined annealing temperature. In some embodiments, once thetemperature is modulated to a temperature above the predeterminedannealing temperature, the temperature is cooled down to thepredetermined annealing temperature at a ramp rate of about 0.1°C./second to about 1.0° C./second (e.g., about 0.1° C/second to about0.9° C./second, about 0.1° C./second to about 0.8° C./second, about 0.1°C/second to about 0.7° C./second, about 0.1° C./second to about 0.6°C./second, about 0.1° C/second to about 0.5° C./second, about 0.1°C./second to about 0.4° C./second, about 0.1° C/second to about 0.3°C./second, about 0.1° C./second to about 0.2° C./second, about 0.2°C/second to about 1.0° C./second, about 0.2° C./second to about 0.9°C./second, about 0.2° C/second to about 0.8° C./second, about 0.2°C./second to about 0.7° C./second, about 0.2° C/second to about 0.6°C./second, about 0.2° C./second to about 0.5° C./second, about 0.2°C/second to about 0.4° C./second, about 0.2° C./second to about 0.3°C./second, about 0.3 to about 1.0° C./second, about 0.3° C./second toabout 0.9° C./second, about 0.3° C./second to about 0.8° C/second, about0.3° C./second to about 0.7° C./second, about 0.3° C./second to about0.6° C/second, about 0.3° C./second to about 0.5° C./second, about 0.3°C./second to about 0.4° C/second, about 0.4° C./second to about 1.0°C./second, about 0.4° C./second to about 0.9° C/second, about 0.4°C./second to about 0.8° C./second, about 0.4° C./second to about 0.7°C/second, about 0.4° C./second to about 0.6° C./second, about 0.4°C./second to about 0.5° C/second, about 0.5° C./second to about 1.0°C./second, about 0.5° C./second to about 0.9° C/second, about 0.5°C./second to about 0.8° C./second, about 0.5° C./second to about 0.7°C/second, about 0.5° C./second to about 0.6° C./second, about 0.6°C./second to about 1.0° C/second, about 0.6° C./second to about 0.9°C./second, about 0.6° C./second to about 0.8° C/second, about 0.6°C./second to about 0.7° C./second, about 0.7° C./second to about 1.0°C/second, about 0.7° C./second to about 0.9° C./second, about 0.7°C./second to about 0.8° C/second, about 0.8° C./second to about 1.0°C./second, about 0.8° C./second to about 0.9° C/second, or about 0.9°C./second to about 1.0° C./second).

In some embodiments, denaturing includes temperature cycling. In someembodiments, denaturing includes alternating between denaturingconditions (e.g., a denaturing temperature) and non-denaturingconditions (e.g., annealing temperature).

(d) Digestion of the Amplified Second Stand and Amplification of theFragments

This disclosure features methods of improving sensitivity of spatialdetection of an analyte in a biological sample where the method includesdigesting (or fragmenting) an amplified second strand to produce secondstrand fragments. In a non-limiting example, the digesting step includesinducing a plurality of double-stranded breaks in an amplified secondstrand. In some embodiments, the double-stranded break occurs at aspecific nucleic acid position. In some embodiments, the second strandfragment includes a sequence of the second strand or a complementthereof and a sequence of the spatial barcode or a complement thereof.

In some embodiments, a double stranded break is induced using arestriction enzyme and a restriction site. In such cases because theproduct of an RCA reaction is a continuous single-stranded molecule(e.g., amplified second strand), a portion of the single-strandedmolecule (e.g., amplified second strand) is made into a double strandedmolecule. In some embodiments, the single-stranded molecule is convertedinto a double stranded molecule by contacting the single-strandedmolecule with an oligonucleotide that is substantially complementary toa portion of the single-stranded molecule (e.g., amplified secondstrand) and includes a sequence that can serve as a restriction site fora restriction enzyme.

In some embodiments, the restriction site is not present in the sequenceof the analyte bound to the capture probes. In such cases, when theamplified second strand is contacted with the oligonucleotide and therestriction enzyme, the oligonucleotide will not hybridize to theportion of the second strand that includes a sequence derived from theanalyte. In some embodiments, the restriction site sequence is presentin the second portion of the second strand.

In some embodiments, the digesting step includes contacting theamplified second strand with an oligonucleotide, where uponhybridization forms a double-stranded sequence with the amplified secondstrand. In some embodiments, the oligonucleotide includes onenucleotide, two nucleotides, three nucleotides, four nucleotides, fivenucleotides, six nucleotides, seven nucleotides, eight nucleotides, ninenucleotides, ten nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, at least 25nucleotides, at least 30 nucleotide, at least 35 nucleotides, at least40 nucleotides, at least 45 nucleotides, or at least 50 nucleotides. Insome embodiments, the oligonucleotide is substantially complementary toa portion of the amplified second strand. By substantiallycomplementary, it is meant that the oligonucleotide is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to asequence in the amplified second strand. In some embodiments, followingdigestion, the residual oligonucleotide that remains hybridized. In someembodiments, following digestion, the oligonucleotide is de-hybridizedfrom the amplified second strand.

In some embodiments, the oligonucleotide includes a 3′ blocking moiety.The 3′ blocking moiety prevents the oligonucleotide from being used as aprimer in an RCA reaction. In some embodiments, the free 3′ end of theoligonucleotide can be blocked by chemical modification, e.g., additionof an azidomethyl group as a chemically reversible capping moiety suchthat the capture probes do not include a free 3′ end. Non-limitingexamples of 3′ modifications include dideoxy C-3′ (3′-ddC), 3′ inverteddT, 3′ C3 spacer, 3′Amino, and 3′ phosphorylation.

In some embodiments where the digesting step includes digesting theamplified second strand with a restriction enzyme, the restrictionenzyme can induce a blunt end or sticky end (e.g., not a blunt end)break. In cases where digestion results in one or more blunt ends, thesecond strand fragments that include the blunt ends can bedephosphorylated (e.g., Shrimp Alkaline Phosphatase (SAP)) so that theends do not reanneal or anneal to another blunt ended second strandfragment.

In some embodiments, the restriction enzyme is a rare-cuttingrestriction endonuclease. A rare-cutting restriction endonucleaseincludes a restriction site about once per 10 kilobases (kb), about onceper 20 kb, about once per 30 kb, about once per 40kb, once per 50 kb,about once per 60 kb, about one per 70 kb, about once per 80 kb, aboutonce per 90 kb, about once per 100kb, about once per 200 kb, about onceper 300 kb, about once per 400 kb, about once per 500 kb, about once per750kb, or about once per 1 megabase (mb) of gDNA. In some embodiments,the restriction enzymes includes restriction enzymes with a 6-bprestriction site or an 8-bp restriction site. Non-limiting examples ofrestriction enzymes that can be used to digest the amplified secondstrand, include Notl, SalI, SfiI, NruI, MluI, SacII, and BssHII. In someembodiment, the restriction enzymes is NotI. In some embodiments, therestriction enzyme is SfiI. In some embodiments, the restriction enzymecan be a non-naturally occurring restriction enzyme.

Non-limiting examples of other methods for inducing a double-strandedDNA break include target specific nucleases (e.g., a CRISPR/Cas systemwhere a Cas nuclease is directed to a specific sequence on the amplifiedsecond strand).

In some embodiments, the method further includes amplifying the secondstrand fragment, thereby generating an amplified second strandfragment(s). In some embodiments, amplifying the second strand fragmentsincludes circle-to-circle amplification as described in Dahl et al.PNAS, 101(13): 4548-4553 (2004), the entire contents of which areincorporated herein by reference. In some embodiments, the methodfurther includes determining the sequence of the amplified second strandfragment, wherein the determined sequence of the amplified second strandfragment includes the spatial barcode sequence of the capture probe or acomplementary sequence thereof, and using the determined sequence of theamplified second strand fragment or the spatial barcode to spatiallydetect the analyte in the biological sample.

(e) Slides, Biological Samples, and Analytes

This disclosure features a method for identifying a location of ananalyte in a biological sample using a substrate (e.g., a firstsubstrate) that includes a plurality of capture probes, where a captureprobe of the plurality of capture probes include a capture domain but nospatial barcode. In some embodiments, the capture probe is affixed tothe substrate at a 5′ end. In some embodiments, the plurality of captureprobes are uniformly distributed on a surface of the substrate. In someembodiments, the plurality of capture probes are located on a surface ofthe substrate but are not distributed on the substrate according to apattern. In some embodiments, the substrate (e.g., a second substrate)includes a plurality of capture probes, where a capture probe of theplurality of capture probes includes a capture domain and a spatialbarcode.

In some embodiments, the capture domain includes a sequence that is atleast partially complementary to the analyte or the analyte derivedmolecule. In some embodiments, the capture domain of the capture probeincludes a poly(T) sequence. In some embodiments, the capture domainincludes a functional domain. In some embodiments, the functional domainincludes a primer sequence. In some embodiments, the capture probeincludes a cleavage domain. In some embodiments, the cleavage domainincludes a cleavable linker from the group consisting of aphotocleavable linker, a UV-cleavable linker, an enzyme-cleavablelinker, or a pH-sensitive cleavable linker.

In some embodiments, the capture domain of the capture probe includes anon-homopolymeric sequence. In some embodiments, the capture domain is adefined sequence. In such cases, the defined sequence can besubstantially complementary to a portion of the amplified padlockoligonucleotide. When the capture probe is substantially complementaryto a portion of the amplified oligonucleotide, the capture domain can beused to capture all or a portion of the padlock oligonucleotide. In someinstances, a defined sequence is about 5 to 50 nucleotides in length(e.g., about 5 to 25, about 5 to 20, about 10 to 25, about 10 to 20). Insome instances, the length of the defined sequence is about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 nucleotides long. Defined sequences can includenatural or synthetic nucleic acids. It is appreciated that a skilledartisan can design the defined sequence in order to capture a particulartarget or particular targets of interest.

In some embodiments, the capture domain of the capture probe includes arandom sequence. In some embodiments, the capture domain includes anon-random sequence. For example, a capture domain with a non-randomsequence can include, without limitation, a defined sequence or ahomopolymeric sequence.

In some embodiments, the biological sample includes a FFPE sample. Insome embodiments, the biological sample includes a tissue section. Insome embodiments, the biological sample includes a fresh frozen sample.In some embodiments, the biological sample includes live cells.

The methods provided herein can be applied to analyte or analyte derivedmolecules including, without limitation, a second strand cDNA molecule(“second strand”). In some embodiments, the analyte or analyte derivedmolecules include RNA and/or DNA.

(f) Compositions and Kits

In some instances, disclosed herein are compositions and kits that areused to carry out the methods described herein. In some embodiments, thekit includes a splint oligonucleotide and a ligase (e.g., a T4 DNAligase (Rnl2), a SplintR ligase, a single stranded DNA ligase, or a T4DNA ligase). In some embodiments, the kit further includes one or moreamplification primers (e.g., two or more primers, three or more primers,four or more primers, five or more primers, six or more primers, sevenor more primers, eight or more primers, nine or more primers, or ten ormore primers) and a polymerase (e.g., a Phi29 DNA polymerase). In someembodiments, the kit further includes an oligonucleotide and arestriction enzyme (e.g., any of the exemplary restriction enzymesdescribed herein).

In some embodiments, the kit further includes a first substrateincluding a plurality of capture probes, wherein a capture probe of theplurality includes and a capture domain, wherein the analyte is capableof hybridizing to the capture domain. It is appreciated that the kit caninclude any of the elements of the substrate, array, or capture probesas described herein.

In some embodiments, a kit used to carry out the methods describedherein includes: a substrate for spatial detection of an analyte; one ormore splint oligonucleotides and a ligase; one or more RCA primers and aPhi29 DNA polymerase; and instructions for performing any of the methodsdescribed herein.

In some embodiments, a kit used to carry out the methods describedherein includes: a substrate for spatial detection of an analyte; one ormore splint oligonucleotides and a ligase; one or more RCA primers and aPhi29 DNA polymerase; one or more oligonucleotides and one or morerestriction enzymes; and instructions for performing any of the methodsdescribed herein.

This disclosure features compositions including the circularized secondstrand, wherein the circularized second strand includes a first portionof the second strand, a portion of the second strand that remainedhybridized to the extended capture probe, a second portion of the secondstrand, and a portion of the backbone sequence of the splintoligonucleotide.

EXAMPLES EXAMPLE 1 Method for Improving Sensitivity of Spatial Detectionof an Analyte

This example provides an exemplary method for improving sensitivity ofspatial detection of an analyte in a biological sample. In anon-limiting example, the method includes: hybridizing the analyte to acapture probe, creating a second strand that is complementary to aportion of the analyte and the extended capture probe, denaturing thesecond strand under conditions where a first portion of the secondstrand and a second portion of the second strand de-hybridize from theextended capture probe; hybridizing a splint oligonucleotide to thepartially denatured second strand and using the split oligonucleotide toligate the first and second portions together, thereby creating acircularized second strand; amplifying the circularized second strand,thereby creating an amplified second strand; and determining all or partof the sequence of the amplified second strand to spatially detect theanalyte in the biological sample.

Briefly, a sample (e.g., an FFPE sample) is decrosslinked to removeformaldehyde crosslinks within the sample thereby releasing the analytesfor spatial detection. The tissue samples are incubated with an HClsolution for 1 minute, repeated twice for a total of 3 minutes.Following HCl incubations, the tissue sections are incubated at 70° C.for 1 hour in TE pH 9.0. TE is removed and the tissues are incubated in1xPBS-Tween for 15 minutes.

FIG. 2 shows exemplary, non-limiting, steps for second strand synthesis.The analyte 201 is hybridized to a capture probe 202 via a capture probebinding domain. A nucleic acid extension reaction (indicated by thedotted line and numeral 203) using a DNA polymerase is used to extendthe capture probe thereby creating an extended capture probe 204. Theanalyte is removed (indicated by the dotted line and numeral 205) fromthe extended capture probe. A second strand synthesis primer 206 ishybridized to the extended capture probe. The extended capture probe isthen copied using the second strand synthesis primer, thereby creating asecond strand 207 that includes a sequence of the analyte or acomplement thereof and the sequence of the spatial barcode or acomplement thereof. The second strand remains hybridized to the extendedcapture probe.

As show in FIG. 3A, the second strand 301 is denatured under conditionswhere a first portion 302 of the second strand and a second portion 303of the second strand de-hybridize from the extended capture probe 304.Denaturing conditions include heating the second strand to 10° C. abovethe predetermined annealing temperature for 5 min and cooling at a rateof 0.5° C/second back down to the predetermined annealing temperature.

The splint oligonucleotide 305 includes a first sequence 306 that issubstantially complementary to the second portion 303, and a secondsequence 307 that is substantially complementary to the first portion302. Following de-hybridizing of the first portion 302 and secondportion 303, a splint oligonucleotide 305 is hybridized to the firstportion 302 and the second portion 303.

The splint oligonucleotide mediates ligation between the second portionand the first portion, thereby creating a circularized second strand.Briefly, a nucleic acid extension reaction 308 is used to extend thesecond portion of the second strand in order to place the second portiondirectly adjacent to the first portion by using the middle section ofthe splint oligonucleotide as the template. The second portion isligated to the first portion, thereby creating a circularized secondstrand 309.

As shown in FIG. 3B, the circularized second strand is amplified tocreate an amplified second strand 310. Amplification includes rollingcircle amplification where the 3′ end of the extended capture probe 311is hybridized to the circularized second strand (e.g., the first portion302 of the second strand included in the circularized second strand).Finally, all or part of the sequence of the amplified second strand issequenced and the sequence is used to determine the spatial location ofthe analyte in the biological sample.

EXAMPLE 2 Digestion of the Amplified Second Strand and Analysis ofSecond Strand Fragments

This example provides an exemplary method for improving sensitivity ofspatial detection of an analyte in a biological sample. In anon-limiting example, the method includes digesting an amplified secondstrand thereby generating second strand fragments. As shown in FIG. 4 ,the amplified second strand 401 is contacted with an oligonucleotide 402where the oligonucleotide includes a sequence that is substantiallycomplementary to a portion of the amplified second strand. Hybridizingthe oligonucleotide to the amplified second strand creates one or moredouble stranded restriction sites in the amplified second strand. Theamplified second strand including the restriction site is contacted witha restriction enzyme that generates double-stranded breaks (as indicatedby the dashed line and numeral 403). The double stranded breaks createrestriction enzyme fragments of the amplified second strand, therebycreating second strand fragments 404. All or part of the sequence of thefragments of the amplified second strand (e.g., the second strandfragments) is determined and used to spatially detect the analyte in thebiological sample.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, section headings, the materials,methods, and examples are illustrative only and not intended to belimiting.

What is claimed is:
 1. A method of determining location of an analyte ina biological sample, the method comprising: (a) hybridizing the analyteto a capture probe on an array, wherein the capture probe comprises aspatial barcode and a capture domain; (b) extending the capture probeusing the analyte as a template, thereby generating an extended captureprobe, and generating a second strand comprising a sequence that iscomplementary to (i) the analyte or a complement thereof and (ii) thespatial barcode or a complement thereof, wherein the second strand isgenerated using a second strand synthesis primer; (c) denaturing thesecond strand from the extended capture probe under conditions wherein a5′ end of the second strand and a 3′ end of the second stranddehybridize from the extended capture probe; (d) hybridizing a splintoligonucleotide both to the 5′ end of the second strand and to the 3′end of the second strand; (e) generating a circularized second strand;(f) amplifying the circularized second strand, thereby creating anamplified second strand; and (g) determining all or part of the sequenceof the amplified second strand to determine the location of the analytein the biological sample.
 2. The method of claim 1, wherein the analytecomprises a capture domain capture sequence that hybridizes to thecapture domain, and wherein the capture domain comprises a poly(T)sequence.
 3. The method of claim 2, wherein the capture domain capturesequence comprises a poly(A) sequence.
 4. The method of claim 1, whereinthe array comprises a plurality of capture probes.
 5. The method ofclaim 1, wherein extending the capture probe and generating a secondstrand utilize a polymerase or reverse transcriptase.
 6. The method ofclaim 1, wherein denaturing comprises increasing the temperature,thereby dehybridizing the 5′ end of the second strand and the 3′ end ofthe second strand from the extended capture probe.
 7. The method ofclaim 1, further comprising, before step (e), extending the 3′ end ofthe second strand using the splint oligonucleotide as a template,thereby creating an extended 3′ end.
 8. The method of claim 7, whereingenerating the circularized second strand comprises ligating theextended 3′ end to the 5′ end using a ligase.
 9. The method of claim 1,wherein amplifying the circularized second strand comprises rollingcircle amplification (RCA) using the circularized second strand as atemplate.
 10. The method of claim 1, wherein the amplified second strandcomprises (i) the spatial barcode or complement thereof and (ii) all orpart of the analyte or a complement thereof.
 11. The method of claim 1,further comprising hybridizing an oligonucleotide to a portion of theamplified second strand, thereby producing a double-stranded sequence,and wherein the double-stranded sequence comprises a restriction site.12. The method of claim 11, further comprising digesting thedouble-stranded sequence using a restriction enzyme.
 13. The method ofclaim 1, wherein the splint oligonucleotide comprises a first sequencethat is substantially complementary to the 5′ end of the second strand,and a second sequence that is substantially complementary to the 3′ endof the second strand.
 14. The method of claim 1, wherein the captureprobe further comprises one or more functional domains, a uniquemolecular identifier, a cleavage domain, and combinations thereof. 15.The method of claim 1, further comprising phosphorylating the 5′ end ofthe splint oligonucleotide prior to the ligation step, and/orphosphorylating the 5′ end of the second strand.
 16. The method of claim1, wherein the amplifying step comprises hybridizing one or moreamplification primers to the circularized second strand, and amplifyingthe circularized second strand with a polymerase.
 17. The method ofclaim 1, wherein the determining step comprises sequencing all or partof the sequence of the amplified second strand to determine the locationof the analyte in the biological sample.
 18. The method of claim 1,wherein the biological sample comprises a formalin-fixed,paraffin-embedded (FFPE) tissue sample; a fresh tissue sample; or afrozen tissue sample.
 19. The method of claim 1, wherein the secondstrand synthesis primer comprises a universal sequence.
 20. The methodof claim 1, wherein the second strand synthesis primer comprises arandom sequence.